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10-28-2010 Bilirubin Present in Diverse Angiosperms Cary Pirone Department of Biological Sciences, Florida International University, [email protected]

Jodie V. Johnson Department of Chemistry, University of Florida

J. Martin E. Quirke Department of Chemistry and Biochemistry, Florida International University

Horacio A. Priestap Department of Biological Sciences, Florida International University

David W. Lee Department of Biological Sciences, Florida International University

Follow this and additional works at: https://digitalcommons.fiu.edu/cas_bio Part of the Biology Commons

Recommended Citation Pirone, C., Johnson, J. V., Quirke, J. M. E., Priestap, H. A., & Lee, D. (March 29, 2010). Bilirubin present in diverse angiosperms. Aob , 2010.

This work is brought to you for free and open access by the College of Arts, Sciences & Education at FIU Digital Commons. It has been accepted for inclusion in Department of Biological Sciences by an authorized administrator of FIU Digital Commons. For more information, please contact [email protected]. AoB Plants Advance Access published October 28, 2010

1

OPEN ACCESS - RESEARCH ARTICLE

Bilirubin Present in Diverse Angiosperms

Cary Pirone1,*, Jodie V. Johnson2, J. Martin E. Quirke3, Horacio A. Priestap1 & David Lee1 Downloaded from

1Department of Biological Sciences, Florida International University, 11200 SW 8 aobpla.oxfordjournals.org St., OE-167, Miami, FL 33199

2Department of Chemistry, University of Florida, P.O. Box 117200, Gainesville,

FL 3261, USA by guest on October 29, 2010

3Department of Chemistry and Biochemistry, Florida International University,

11200 SW 8 St., CP-304, Miami, FL, 33199, USA

*Corresponding authors’ e-mail address: Cary Pirone: [email protected]

Received: 20 August 2010; Returned for revision: 25 September 2010 and 22 October 2010; Accepted: 24 October 2010

© The Author 2010. Published by Oxford University Press. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non- Commercial License (http://creativecommons.org/licenses/by-nc/2.5), which permits unrestricted non- commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. 2

ABSTRACT

Background and aims: Bilirubin is an orange-yellow tetrapyrrole produced from

the breakdown of heme by mammals and some other vertebrates. Plants,

algae, and cyanobacteria synthesize molecules similar to bilirubin, including the

protein-bound bilins and phytochromobilin which harvest or sense light.

Recently, we discovered bilirubin in the arils of nicolai, the White Bird of

Paradise , which was the first example of this molecule in a higher . Downloaded from Subsequently, we identified bilirubin in both the arils and of Strelitzia

reginae, the Bird of Paradise . In the arils of both , bilirubin is

present as the primary pigment, and thus functions to produce color. Previously, aobpla.oxfordjournals.org

no tetrapyrroles were known to generate display color in plants. We were

therefore interested in determining whether bilirubin is broadly distributed in the

plant kingdom, and whether it contributes to color in other species. by guest on October 29, 2010

Methodology: In this paper, we use we use HPLC/UV and

HPLC/UV/electrospray ionization-tandem mass spectrometry (HPLC/UV/ESI-

MS/MS) to search for bilirubin in ten species across diverse angiosperm lineages.

Principal results: Bilirubin was present in eight species from the orders

Zingiberales, Arecales, and , but only contributed to color in species

within the .

Conclusions: The wide distribution of bilirubin in angiosperms indicates the need to re-assess some metabolic details of an important and universal biosynthetic pathway in plants, and further explore its evolutionary history and 3

function. Although color production was limited to the Strelitziaceae in this study,

further sampling may indicate otherwise.

INTRODUCTION

Tetrapyrroles occur throughout the plant kingdom; this class of molecules

includes vital biosynthetic products such as chlorophyll and heme. In plants, the

degradation of heme forms first biliverdin IX- α, and subsequently Downloaded from phytochromobilin, the precursor of the phytochrome chromophore, an essential

light-sensing molecule (Tanaka et al., 2007). In mammals and some vertebrates,

biliverdin-IXα is also formed from the degradation of heme, but it is transformed aobpla.oxfordjournals.org into the yellow-orange pigment bilirubin-IX α. We have identified bilirubin-IXα

(henceforth referred to as bilirubin) as the major pigment in the orange arils of

Strelitzia nicolai, the White Bird of Paradise Tree (Pirone et al., 2009). Although by guest on October 29, 2010 ubiquitous in animals, this is the first example of bilirubin in a plant.

Subsequently, we have discovered this pigment in the and arils of S. reginae, the bird of paradise flower, indicating the pigment is not unique to S.

nicolai (Pirone et al., in press).

In S. nicolai and S. reginae, bilirubin is a novel biosynthetic source of

display color. As a rule, the coloration of flowers and is achieved with

products from three metabolic pathways: the terpenoid (carotenoids), the

phenylpropanoid (flavonoids), and the betalain (betalains) (Davies, 2004;

Grotewold, 2006; Lee, 2007). Betalain synthesis is restricted to families in the

order Caryophyllales, while carotenoids and flavonoids (including anthocyanins) 4 are pervasive in the plant kingdom (Harbourne, 1967; Goodwin, 1988). A rare group of pigments, the phenalenones, has been documented in several species in the Strelitziaceae and related families (Davies, 2004). However, to our knowledge, neither the phenalenones nor other rare pigments play a significant role in color production. Bilirubin is thus the first product of an additional biosynthetic route, the tetrapyrrole pathway, to produce conspicuous color in a plant reproductive structure. Chlorophylls, which are also synthesized via the Downloaded from tetrapyrrole pathway, primarily produce color in foliage, thus forming a green background upon which the contrasting colors of flowers and fruits are displayed.

While chlorophylls occasionally produce color in reproductive structures, these aobpla.oxfordjournals.org are fairly inconspicuous.

Given the presence of bilirubin in Strelitzia, it is interesting to determine if the pigment is produced by other taxa within the Strelitziaceae, in families by guest on October 29, 2010 closely allied to the Strelitziaceae (as in the ), as well as throughout the major groups of the angiosperms. Preliminary high performance liquid chromatography (HPLC/UV) analyses of aril extracts of an additional species in the Strelitziaceae, Phenakospermum guyanense, showed a pigment with a retention time and UV-Visible spectra which matched those of bilirubin. Here, we use HPLC/UV and HPLC/UV/electrospray ionization-tandem mass spectrometry

(HPLC/UV/ESI-MS/MS) to confirm the presence of bilirubin in P. guyanense and investigate the presence of bilirubin in the mature fruits from nine additional species and the flowers of a single additional species. Six species are within the order Zingiberales, and four are from diverse angiosperm orders (Table 1). We 5

discuss our findings within a phylogenetic and biochemical context, and comment

on a possible ecological role for bilirubin as a color signal to attract animal

dispersers and pollinators.

MATERIALS AND METHODS

Plant material was collected from Fairchild Tropical Botanic Garden in Miami, FL

except aril tissue from Strelitzia reginae, which was obtained from Ellison Downloaded from Horticulture Pty. Ltd. in Allstonville, New South Wales, Australia. Tissue for each

sample and its replicate were composed of tissue from one or multiple

or infructescences from a single, sometimes clonal, individual. aobpla.oxfordjournals.org

The replicate aril samples of Phenakospermum guyanense came from different

individuals (collected by John Kress; Guyana (South America), Demerara-

Mahaica region). For the names and taxonomic affiliations of species sampled, by guest on October 29, 2010

see Table 1. We sampled species from each banana group family, except from

the Lowiaceae. This monotypic family consists of fifteen rare species within

Orchidantha, and we were not able to obtain enough material for analysis. We

selected balbisiana (Musaceae), one of the wild progenitors of most

cultivated bananas (Heslop-Harrison and Schwarzacher, 2007),

collinsiana (Heliconiaceae), and representatives from each of the two

Strelitziaceae genera not previously analyzed for bilirubin content,

Phenakospermum guyanense and madagascariensis. We also sampled species from two of the most derived families in the order, Costus lucanusianus () and Hedychium coronarium (Zingiberaceae) (Kress, 6

2001). We mainly sampled orange fruits to maximize the potential chances of

finding bilirubin, but we also included the blue arils of Ravenala

madagascariensis, the yellow fruits of Heliconia collinsiana, and the multi-colored

flowers of Costus lucanusianus. To determine whether bilirubin is present in

plants outside of the Zingiberales, we sampled species from the basal dicot order

Laurales, two monocot orders, the Arecales and the Pandanales, and the eudicot

order Myrtales, which is part of the Rosid clade. Selection of species within Downloaded from those orders (Table 1) was based on tissue availability and color. For each

sample (except aril samples), 20.0 g of fresh tissue was ground in a blender with

100 mL methanol for two minutes, and was then filtered through a Buchner aobpla.oxfordjournals.org

funnel. The residue was re-extracted with chloroform in a mortar and pestle.

Methanol and chloroform extracts were pooled, and 100 mL of water was added.

The mixture was left in a separatory funnel for five minutes, and then the (lower) by guest on October 29, 2010

chloroform layer was collected, filtered with a polytetrafluoroethelene (PTFE) 0.2

µm filter, and divided into two equal aliquots. Each aliquot was dried to completion in a rotovap at 30 C. For each aril sample, 0.05 g tissue from a single aril was ground by a mortar and pestle and extracted with chloroform repeatedly until the chloroform extracts were colorless. As above, the chloroform extract was filtered, divided into two equal aliquots, and dried on a rotovap. All tissues were sampled in duplicate. To determine the presence of bilirubin, one aliquot

from each sample was analyzed via HPLC and the second aliquot was analyzed

via HPLC/ESI-MS/MS.

7

HPLC/UV

HPLC/UV analyses were performed on a Thermo-Finnigan SpectraSystem HPLC

apparatus with a variable wavelength photodiode array (PDA) detector

(SMC1000, P4000, AS3000, UV6000LP; Thermo Electro Corporation, San Jose,

CA, USA). Extract was redissolved in DMSO, partitioned with hexane to remove lipids, and chromatographed on a reverse phase ODS-A column (150 mm × 4.3 mm, particle size 5µm; Waters, Milford, MA, USA). Mobile phase A was 0.1% Downloaded from formic acid in methanol, and mobile phase B was 0.1% formic acid in water. The

HPLC gradient (at 1.0 mL/min) was started at 40%A and increased linearily to

95%A over 40 min, then held constant at 95%A and 5%B for 10 minutes. aobpla.oxfordjournals.org

Bilirubin was identified by comparing the retention time and UV-Visible spectra of

sample pigments with bilirubin standard (Sigma-Aldrich; St. Louis, MO, USA),

which had a retention time of 42.9 min and a maximum absorbance at 444 nm in by guest on October 29, 2010

the above HPLC solvent system. Bilirubin concentrations were determined by

comparison with a standard curve [(R2 = .995) estimated detection limit = 20 ng

injected on column]. Preliminary analysis of some plant extracts showed

compounds which eluted at retention times similar to that of bilirubin. The UV-

Visible spectra of these compounds were similar to carotenoids. To avoid the possible overlap of the HPLC/UV spectra of these pigments with that of bilirubin, we treated non-arillate samples (Table 1) with diazomethane to convert bilirubin to its di-methyl ester, i.e. both carboxylic acids were converted to methyl esters

(λmax= 453 nm in HPLC solvents described above) (Kuenzle, 1973).

Diazomethane was prepared from diazald according to Vogel et al. (1989). 8

Chloroform extracts of the sepals were treated with an excess of a solution of

diazomethane in order to form the bilirubin di-methyl ester. The addition of the

diazomethane was deemed to be complete when effervescence was no longer

observed. The excess diazomethane was destroyed by the addition of a few

drops of acetic acid. Although the diazomethane would also methylate any other

carboxylic acid impurities in the extract, such ester byproducts did not interfere in

any way with the observance of the bilirubin di-methyl ester peak in the HPLC Downloaded from analyses. Thus, it was unnecessary to carry out additional purification of the

extracts. With the HPLC/UV conditions described above, the retention time

of bilirubin di-methyl ester was 31.7 min, thus making it possible to observe the aobpla.oxfordjournals.org

compound without interference from other pigments (Fig. 1). A standard curve for

bilirubin di-methyl ester [(R2 =1), estimated detection limit = 35.0 ng injected on column] was constructed by treating bilirubin standard with diazomethane. by guest on October 29, 2010

Identification of the peak at 31.7 minutes as bilirubin di-methyl ester was verified

by comparison with bilirubin di-methyl ester standard (Frontier Scientific, Logan,

UT, USA), which also eluted at 31.7 minutes. For samples in which bilirubin was

detected via HPLC/ESI-MS/MS but not HPLC/UV, we assumed the mass of

bilirubin to be less than the estimated detection limit of bilirubin or bilirubin

treated with diazomethane. Standard deviation values were not calculated owing

to the low sample size. Instead, concentration values for the replicate samples of

each plant are presented in Table 1.

9

HPLC/UV/ESI-MS/MS

The plant extract was redissolved in DMSO (Certified ACS; Fisher Scientific) and

analyzed via reverse phase C8 HPLC/UV/ESI-MS/MS utilizing both positive and

negative ESI and a number of different MSn scans. HPLC/UV was performed

with an Agilent Technologies HPLC with binary pumps (1100 series; Santa Clara,

CA, USA), a Symmetry C8 HPLC column (150 mm x 2.1 mm, particle size 5µm;

Waters, Milford, MA, USA) and an Agilent UV-Visible detector (G1314A). The Downloaded from mobile phase A was 0.2% acetic acid (glacial, biochemical grade (99.8%);

ACROS organics, Morris Plains, NJ, USA) in H2O (HPLC grade, Honeywell

Burdick & Jackson, Muskegon, MI, USA) and mobile phase B was 0.2% acetic aobpla.oxfordjournals.org

acid in acetonitrile (LC-MS grade, Honeywell Burdick & Jackson). The HPLC

gradient (at 0.2 mL/min) was started at 20%B at time 0 and increased linearily to

85%B over 30 min and then increased linearly to 100%B over 15 min. The by guest on October 29, 2010

column was held at 100%B until monitoring of the UV/MS signal showed no

further elution of peaks. For some extracts this was more than 150 min. The

UV-Vis response was monitored at 450 nm for bilirubin, which eluted at

approximately 39 min.

All mass spectrometry data was obtained with a Finnigan MAT (San Jose, CA,

USA) LCQ classic quadrupole ion trap mass spectrometer equipped with an

electrospray ionization source (ESI). The ESI was operated with a nitrogen

sheath and auxiliary gas flows of 65 and 5, respectively, (unitless instrument parameters) with a spray voltage of 3.3 kV and a heated capillary temperature of 10

250 C. The heated capillary voltage was +15 V and -22 V for (+) and (-)ESI,

respectively, while the tube lens was operated at 0 V for both ESI polarities.

Collision-induced dissociation (CID) was conducted with a parent ion isolation of

3u, CID energy of 37.5%, qCID of 0.25 and CID time of 30 ms.

With (+)ESI, bilirubin produced an m/z 585 [M+H]+ ion and an m/z 583 ion due to

oxidation during ionization. The m/z 585 and m/z 583 ions underwent CID- Downloaded from MS/MS to form m/z 299, and 297 ions, respectively, as major product ions. With

(-)ESI-MS, bilirubin produced m/z 583 [M-H]- and m/z 581 ions which were

dissociated to form m/z 285 and 537 major product ions, respectively. Bilirubin aobpla.oxfordjournals.org

was identified in the plant extracts by matching of retention time and (+) and (-)

ESI-MS and –MS/MS spectra with those of the authentic bilirubin standard.

False positives due to contamination during handling and analyses were avoided by guest on October 29, 2010

by the washing of glassware multiple times (soap and water, acetone,

chloroform), the use of disposable vials when possible, and changing gloves after

the preparation of each sample. Dimethyl sulfoxide (DMSO) solvent blanks were analyzed before and after analyses of each extract and after analyses of bilirubin standards to check for carryover of bilirubin. The DMSO blanks were repeated if significant levels of bilirubin were carried over to subsequent runs.

11

RESULTS

Bilirubin was present in eight of the ten species tested: Musa balbisiana,

Heliconia collinsiana, Costus lucanusianus, Ravenala madagascariensis,

Phenakospermum guyanense, Hedychium coronarium, Gastrococcus crispa, and

Eugenia luschnathiana (Table 1). Bilirubin was present in the fruits (including aril and peel) of all species except Costus lucanusianus, where it was present in the

flowers (fruits were unavailable for sampling). In Ravenala madagascariensis Downloaded from and Phenakospermum guyanense, HPLC/UV analysis of the aril extracts showed

a single peak with retention time and UV-Visible spectrum which matched those

of the bilirubin standard. Bilirubin identification was confirmed by HPLC/ESI- aobpla.oxfordjournals.org

MS/MS. Bilirubin was also identified in Musa balbisiana, Heliconia collinsiana,

Costus lucanusianus, Hedychium coronarium, Gastrococcus crispa, and

luschnathiana via HPLC/ESI-MS/MS, but was not detected via HPLC/UV, even by guest on October 29, 2010

after treatment with diazomethane. In Heliconia collinsiana and Eugenia

luschnathiana, bilirubin was detected in only one of the two replicate samples,

while in all other species bilirubin was detected in both replicates. The

concentration of bilirubin was highly variable among species. Concentrations ranged from less than 44 ng/g of fresh tissue (n=2) to 3.73 mg/g of fresh tissue

(n=2) (Table 1).

12

DISCUSSION

We initially discovered bilirubin in S. nicolai (family: Strelitziaceae, order:

Zingiberales), and also in the arils and sepals of S. reginae. Since similar secondary metabolites are expected to be found within members of a clade, we concentrated most of our sampling within the Strelitziaceae and the Zingiberales.

Kress et al. (2001) divides the order into two major clades, the basal “banana

group” which includes the Musaceae, Strelitziaceae, Heliconiaceae, and Downloaded from Lowiaceae, and the more derived “ginger group”, which includes the four

remaining families (Fig. 2). Although resolution of intrafamilial relationships

varies among other studies, the ginger clade families are generally well resolved aobpla.oxfordjournals.org

(Rudall et al., 1999; Chase et al., 2000; Givnish, 2006), and there is high support

for the position of the Lowiaceae as sister to the Strelitziaceae (Rudall et al.,

1997; Chase et al., 2000; Soltis et al., 2000; Givnish et al., 2006; Soltis et al., by guest on October 29, 2010

2007).

The detection of bilirubin in Gastrococos crispa (family: Arecaceae, order:

Arecales) and Eugenia luschnathiana (family: , order: Myrtales),

indicates that bilirubin is not restricted to the Zingiberales and may be broadly

distributed throughout the plant kingdom. However, the lack of detection of

bilirubin in avocado, Persea americana (family: Lauraceae, order: Laurales) and

Pandanus odoratissimus (family: Pandanaceae, order: Pandanales) suggests

that bilirubin is not universal in plants at levels detectable by mass spectrometry.

13

The high concentration of bilirubin in the arils of P. guyanense indicates its role in color production. Previous studies indicated bilirubin is also responsible for color production in two other Strelitziaceae species, S. nicolai and S. reginae. Since brightly colored fruit displays often serve as signals to attract dispersers (van der

Pijl, 1982), it is likely that in these species, bilirubin contributes to the attraction of avian frugivores which feed upon the arils (Frost, 1980; Kress, personal communication). Whether bilirubin plays an additional role beyond color Downloaded from production in plants remains to be determined. Bilirubin may function as a potent antioxidant in plants as it does in humans (Stocker, 1987), or serve a different

physiological function. Bilirubin may also be a mere metabolic waste product. aobpla.oxfordjournals.org

The detection of bilirubin in only one replicate of H. collinsiana and E. luschnathiana, and the variability of bilirubin concentration within P. guyanense by guest on October 29, 2010 may indicate that bilirubin biosynthesis is not constant, but is instead variable and influenced by factors which are currently unknown. For example, we observed the accumulation of bilirubin in aril cells during the development of S. nicolai

(personal observation), with a maximum quantity present in mature tissue (Fig.

3), suggesting that bilirubin production may be influenced by development. The lack of bilirubin at the time of sampling may also be a function of variable production, and thus may not indicate an absence of bilirubin biosynthesis in the species.

14

The biochemical pathway which produces bilirubin in plants remains unknown.

In animals, biliverdin-IX α is reduced by an NAD(P)H-dependent biliverdin-IX α

reductase (BR) to form bilirubin-IX α (Maines and Trakshel, 1993). Conversely,

in plants, biliverdin-IX α is converted to a structural isomer of bilirubin, 3Z- phytochromobilin, the precursor of the phytochrome chromophore (Tanaka and

Tanaka, 2007), by a ferredoxin-dependent bilin reductase, phytochromobilin synthase (Terry et al., 1995; Kohchi et al., 2001). In cyanobacteria and some Downloaded from algae, biliverdin-IX α is also reduced by ferredoxin-dependent enzymes, the bilin reductases, to form the phycobilin chromophores (Beale, 1993; Frankenburg et

al., 2001). A BR enzyme was identified and cloned in the cyanobacterium aobpla.oxfordjournals.org

Synechocystis (Schluchter and Glazer, 1997), and BR enzymes have also been found in a variety of other bacteria. Whether bilirubin IX-α in plants is formed by

the reduction of biliverdin IX-α by a BR enzyme or via some other means remains by guest on October 29, 2010

to be determined.

CONCLUSIONS and FORWARD LOOK

Bilirubin was present in eight species from three diverse angiosperm orders, and

contributed to aril color in species within the Strelitziaceae. Further sampling of

bilirubin in plants, both across species, under variable conditions, and across

different time scales, will be necessary to gain a comprehensive understanding of

the distribution of bilirubin in plants, and to determine whether color production is limited to the Strelitziaceae. These studies, combined with work on the 15

biosynthesis of bilirubin in plants, will provide a more comprehensive

understanding of the evolution of this unusual molecule in the plant kingdom.

SOURCES OF FUNDING

This research was funded by the United States Environmental Protection Agency

(EPA) under the Greater Research Opportunities (GRO) Graduate Program and

the Office of Research (OSRA) at FIU. Funding for the Finnigan MAT LCQ Downloaded from Classic was provided by NSF CHE 9613678. We thank Waters Corporation for

the donation of the C8 HPLC column.

aobpla.oxfordjournals.org

CONTRIBUTIONS BY AUTHORS

XXX wrote the manuscript, collected plants, prepared extracts, and performed

HPLC analyses. XXX performed and interpreted MS/MS analyses. XXX also by guest on October 29, 2010 interpreted MS/MS spectra and oversaw lab work. XXX prepared the Hedychium

samples and provided general lab advice. XXX was the first to observe the

uniqueness of Strelitziaceae pigments, and contributed to the discussion on

ecology and color production.

CONFLICTS OF INTEREST

No conflicts of interest 16

ACKNOWLEDGEMENTS

We thank the Center for Ethnobiology and Natural Products (CeNAP) at Florida

International University for laboratory facilities, K. G. Furton for his generous

support of H.A. Priestap, and J. Kress for Phenakospermum material.

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by guest on October 29, 2010 20

FIGURE LEGENDS

Figure 1. HPLC/UV Chromatograms of A. Bilirubin standard, B. Bilirubin standard treated with diazomethane, and C. Carotenoid-like pigments from the flowers of Costus lucanusianus, which elute around the same retention time as bilirubin. Bilirubin standard and flower extract from C. lucanusianus monitored at 444 nm, bilirubin standard treated with diazomethane monitored at

453 nm. Downloaded from

Figure 2. Phylogeny of the Zingiberales. Phylogenetic relationships of genera in the Zingiberales (figure adapted from Kress et al., 2001) with photographs of aobpla.oxfordjournals.org four species sampled in this study.

Figure 3. Bilirubin production in Strelitzia nicolai during aril development.

Maturing arils of S. nicolai with increasing amounts of bilirubin. ~4 mm in diameter. by guest on October 29, 2010

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Fig. 1 22

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Fig. 2 23

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Fig. 3 aobpla.oxfordjournals.org

by guest on October 29, 2010 Table 1. Summary of HPLC/UV and MS/MS results of the analysis of bilirubin (BR) in ten angiosperm species. N/A indicates that samples were not treated with diazomethane.

Species Family Order Organ BR BR BR BR BR Mean detection detectio detectio conc. conc. BR via n n via Samp. Samp. conc. Diazo- via HPLC- 1 2 (n=2) methane HPLC/ MS/MS Deriv- UV ative Musa Musaceae Zingiberales peel N N Y < 44 < 44 ― balbisiana ng/g ng/g

Heliconia Heliconiaceae Zingiberales fruit N N Y ― < 44 ― Downloaded from collinsiana ng/g

Costus Costaceae Zingiberales flower N N Y < 44 < 44 ― luca- ng/g ng/g nusianus

aobpla.oxfordjournals.org Ravenala Strelitziaceae Zingiberales aril N/A Y Y 0.001 0.001 0.001 madagas- mg/g mg/g mg/g cariensis

Phenako- Strelitziaceae Zingiberales aril N/A Y Y 3.041 5.787 3.725 spermum mg/g mg/g mg/g guyanense

Hedychium Zingiberaceae Zingiberales aril N N Y < 44 < 44 ― by guest on October 29, 2010 coronarum ng/g ng/g

Gastro- Arecaceae Arecales fruit N N Y < 44 < 44 ― cocos ng/g ng/g crispa

Pandanus Pandanaceae Pandanales fruit N N N ― ― ― odora- tissimus

Persea Lauraceae Laurales fruit N N N ― ― ― americana

Eugenia Myrtaceae Myrtales fruit N N Y < 44 ― ― lusch- ng/g nathiana